Neuromodulation techniques for perturbation of physiological systems

ABSTRACT

Embodiments of the present disclosure relate to techniques for inducing physiological perturbations in a subject via neuromodulation, e.g., peripheral neuromodulation of a region of interest of an organ. The nature and degree of the perturbations may be related to the subject&#39;s clinical condition. Accordingly, an assessment of one or more characteristics of the perturbations may be used to determine a clinical condition of the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 16/399,692, filed on Apr. 30, 2019, the disclosure of which isincorporated by reference herein for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under contract numberHR0011-18-C-0040 awarded by the Defense Advanced Research ProjectsAgency (DARPA). The government has certain rights in the invention.

BACKGROUND

The subject matter disclosed herein relates to techniques to assessphysiological systems via intentional perturbation of such systems andassessment of recovery from and/or characteristics of the perturbation.In particular, the perturbation may be the result of targetedneuromodulation.

Healthcare providers use a variety of methods to test for and diagnoseclinical conditions. In certain cases, a subject may be subjected toconditions that induce a stress or perturbation of some kind, and anassessment of the subject's response to the stress or perturbation maybe indicative of a clinical condition or overall health. Suchperturbations may be induced by exercise, pharmacologic agents, fasting,etc. For example, a glucose tolerance test may involve administering aglucose load to a subject and assessing blood samples taken from thesubject after the administration to determine how the subject responds.However, such testing is complex and may require dietary restrictions(e.g., fasting in advance) with which some subjects may not comply,which in turn may influence the results. Further, administration ofpharmacologic agents may involve undesirable side effects. Accordingly,improved techniques for diagnosing certain clinical conditions would bebeneficial.

BRIEF DESCRIPTION

The disclosed embodiments are not intended to limit the scope of theclaimed subject matter, but rather these embodiments are intended onlyto provide a brief summary of possible embodiments. Indeed, thedisclosure may encompass a variety of forms that may be similar to ordifferent from the embodiments set forth below.

In one embodiment, a modulation system is provided that includes anenergy application device configured to apply energy to a region ofinterest to cause a physiological perturbation in a subject. The systemalso includes a controller configured to control application of theenergy via the energy application device to the region of interest toinduce the physiological perturbation to cause a change in concentrationof one or more molecules of interest relative to a baselineconcentration. The controller is also configured to receive informationindicative of the concentration of the one or more molecules of interestand determine that the subject is in is in category selected from two ormore categories based on a change in the concentration of the one ormore molecules of interest relative to the baseline concentration withina time period.

In another embodiment, a method of inducing a physiological perturbationin a subject is provided. The method includes the steps of directing anenergy application device at a region of interest of a subject; applyingthe energy to the region of interest to modulate activity of at leastone axon terminal within the region of interest as a result of theapplying; assessing one or more characteristics of the perturbation atone or more time points after the perturbation; and providing anindication of a clinical condition of the subject based on theassessing.

In another embodiment, a modulation system is provided that includes anenergy application device configured to apply energy to a first regionof interest in a first organ and to a second region of interest in asecond organ. The system also includes a controller configured tocontrol a first application of the energy via the energy applicationdevice to the first region of interest to cause a first perturbation asa result of the first application of the energy; receive informationindicative of a first perturbation characteristic; control a secondapplication of the energy via the energy application device to thesecond region of interest to cause a second perturbation as a result ofthe second application of the energy; receive information indicative ofa second perturbation characteristic; determine a clinical condition ofthe subject based on the first perturbation characteristic and thesecond perturbation characteristic; and provide an indication of theclinical condition.

In another embodiment, a method of assessing a physiologicalperturbation in a subject is provided. The method includes the steps ofapplying ultrasound energy to a region of interest in a subject to causean approximated fasting state in the subject via neuromodulation;receiving glucose and insulin concentration data from the subject in theapproximated fasting state; applying the data to a model, wherein themodel is based on a relationship between glucose and insulinconcentration in the approximated fasting state for a plurality ofnormal subjects; receiving an indication of an insulin resistance of thesubject using the model; and providing a treatment recommendation basedon the indication.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 is a schematic representation of a neuromodulation system using apulse generator according to embodiments of the disclosure;

FIG. 2 is a block diagram of a neuromodulation system according toembodiments of the disclosure;

FIG. 3 is a schematic representation of a neuromodulation

FIG. 4 is a flow diagram of a neuromodulation technique according toembodiments of the disclosure;

FIG. 5 is a flow diagram of a neuromodulation technique according toembodiments of the disclosure;

FIG. 6 is a flow diagram of a neuromodulation technique according toembodiments of the disclosure;

FIG. 7 is a schematic representation of an ultrasound energy applicationdevice in operation according to embodiments of the disclosure;

FIG. 8 is a schematic illustration of the experimental setup forultrasound energy application to achieve targeted physiologicalperturbations according to embodiments of the disclosure;

FIG. 9 is an experimental timeline of ultrasound energy applicationaccording to embodiments of the disclosure;

FIG. 10 shows the circulating glucose concentration after ultrasoundenergy application in a Zucker-HFD animal type 2 diabetes modelaccording to embodiments of the disclosure;

FIG. 11 shows food consumption in the animals during the experimentaltimeline of FIG. 10;

FIG. 12 shows a change in rate of body weight gain in treated vs.control animals in the animals during the experimental timeline of FIG.10;

FIG. 13 shows modified Homeostatic Model Assessment of InsulinResistance scores using a model based on neuromodulation techniqueaccording to embodiments of the disclosure in the animals during theexperimental timeline of FIG. 10;

FIG. 14 is a flow diagram of a neuromodulation technique according toembodiments of the disclosure;

FIG. 15 shows a profile of a response and recovery to a neuromodulationperturbation according to embodiments of the disclosure in a highlyinsulin resistant subject;

FIG. 16 shows a profile of a response and recovery to a neuromodulationperturbation according to embodiments of the disclosure in a lessinsulin resistant subject;

FIG. 17 shows a profile of a response and recovery to a neuromodulationperturbation according to embodiments of the disclosure in a lessinsulin resistant subject;

FIG. 18 is a flow diagram of a neuromodulation technique according toembodiments of the disclosure;

FIG. 19 shows TNF mRNA expression in treated vs. control groups afterLPS exposure;

FIG. 20 shows mRNA expression for various markers in treated vs. controlgroups after LPS exposure; and

FIG. 21 shows an example protocol for acquiring baseline and potentialpost-exposure data for assessing a change in response based toneuromodulation after exposure to a material or pathogen.

DETAILED DESCRIPTION

One or more specific embodiments will be described below. In an effortto provide a concise description of these embodiments, not all featuresof an actual implementation are described in the specification. Itshould be appreciated that in the development of any such actualimplementation, as in any engineering or design project, numerousimplementation-specific decisions must be made to achieve thedevelopers' specific goals, such as compliance with system-related andbusiness-related constraints, which may vary from one implementation toanother. Moreover, it should be appreciated that such a developmenteffort might be complex and time consuming, but would nevertheless be aroutine undertaking of design, fabrication, and manufacture for those ofordinary skill having the benefit of this disclosure.

Any examples or illustrations given herein are not to be regarded in anyway as restrictions on, limits to, or express definitions of, any termor terms with which they are utilized. Instead, these examples orillustrations are to be regarded as being described with respect tovarious particular embodiments and as illustrative only. Those ofordinary skill in the art will appreciate that any term or terms withwhich these examples or illustrations are utilized will encompass otherembodiments that may or may not be given therewith or elsewhere in thespecification and all such embodiments are intended to be includedwithin the scope of that term or terms. Language designating suchnon-limiting examples and illustrations includes, but is not limited to:“for example,” “for instance,” “such as,” “e.g.,” “including,” “incertain embodiments”, “in some embodiments”, and “in one (an)embodiment.”

Provided herein are techniques for physiological perturbation vianeuromodulation of targeted regions of interest and to cause targetedphysiological perturbations that are the result of the modulation. Suchphysiological perturbations may be useful for assessing subjectresponses to the perturbations in a controlled environment. For example,a physiological perturbation may cause metabolic pathways to adjust tothe effects of the perturbation to achieve homeostasis. The quality oreffectiveness of the response may be a measure of a subject's overallcondition. In one embodiment, neuromodulation is used alternatively oradditionally relative to other techniques for inducing physiologicalchanges (e.g., exercise stress tests, pharmacologic agentadministration). While pharmacologic agents may perturb physiologicalsystems, their effects may vary from subject to subject due tovariability in patient comorbidities or variability in physiologicalprocesses that respond to the agents and that are unconnected to thesystems being examined. For example, certain subjects may metabolizepharmacologic agents at different rates due to pharmacogeneticdifferences in drug metabolism pathways. Differences in metabolism ratesmay lead to differences in agent effects. Accordingly, such tests usingpharmacologic agents may fail to identify patients with certain clinicalconditions. That is, certain patients may have responses that aredifficult to fit to existing indices or models of response types.

Neuromodulation applied to cause physiological perturbations may betargeted, allowing healthcare providers to induce desired changes andassess one or more characteristics of the perturbation. Suchcharacteristics may include a change in concentration of one or moremolecules of interest in response to the perturbation and a time torecovery (e.g., an assessment of the persistence of the physiologicalperturbation). Accordingly, the assessed characteristic of theperturbation may be a change in concentration of one or more moleculesof interest over a predetermined time period and/or a total time toachieve a predetermined baseline concentration of one or more moleculeof interest. In certain embodiments, based on the assessment, adiagnosis of a clinical condition may be made and/or treatment ortreatment recommendation to the subject may be provided.

Neuromodulation of regions of interest may cause physiologicalperturbations in healthy or diseased subjects. In one embodiment,neuromodulation that does not cause a characteristic change in a subjectmay be associated with a clinical condition or a disease diagnosis. Inanother embodiments, different clinical conditions or categories may beassociated with certain characteristic responses to the neuromodulation.That is, application of energy to a region of interest and the absenceof predicted physiological responses to the perturbation may beindicative of diseased organs, including metabolic orinflammatory/anti-inflammatory pathways that are unable to respond toneuromodulation in a manner of a healthy patient. In one example,neuromodulating energy applied to pancreas with no resultant increase incirculating insulin levels is indicative of a nonresponsive subject whocan no longer produce insulin. Therefore, a treatment recommendation maybe provided that neuromodulation therapy will not work and insulinshould be provided.

While perturbations may be induced by challenges such as exercise,administration of pharmacologic agents, and/or dietary changes(fasting), such perturbations are typically diffuse and untargeted. Incontrast, as provided herein, neuromodulation may be targeted to one ormore specific structures in the body to cause targeted or predictablephysiological perturbations. Further, insofar as multiple organs may beinvolved in metabolic processes, neuromodulation of one or more regionsof interest in specific organs may be beneficial for identifying partsof a pathway that may be dysfunctional.

As provided herein, neuromodulation may induce physiologicalperturbation via modulation of activity at axon terminals within theregion of interest. The targeted region or regions of interest may beany tissue or structure in the body having axon terminals formingsynapses with non-neuronal cells or fluids. In one example, the regionof interest may be in an organ or structure, such as a spleen, liver,pancreas, or gastrointestinal tissue. In another example, the regions ofinterest may be in a lymph system tissue. Neuromodulation of regions ofinterest permits a local, limited, and nonablative application of energyto only the targeted regions of interest and without the energy beingapplied outside of the regions of interest. Energy application maytrigger efferent perturbations outside of the targeted regions ofinterest, e.g., in the same organ, tissue or structure containing theregion of interest or in other organs and structures that do not containthe targeted region of interest. In some embodiments, the afferenteffects may be induced in areas of a hypothalamus by way of example. Theenergy application may also induce afferent effects along the targetednerve upstream from the site of the energy application. In someembodiments, the effects outside of the targeted region/s of interestmay be achieved without direct energy application to areas outside ofthe region/s of interest where the effects are induced. Accordingly,local energy application may be used to realize or achieve systemicperturbations which may include local effects, downstream effects and/orupstream effects.

The disclosed techniques may be used to exert perturbations ofphysiological processes of the body using an external or extracorporealsource to cause targeted physiological perturbations in subjects. Vianeuromodulation to the targeted regions of interest, physiologicalprocesses may be altered, slowed, halted, or reversed. Also providedherein are techniques that may be applied to subjects to upset ortemporarily challenge the dynamic equilibrium or homeostasis ofphysiological processes, such as glucose regulation; or to stimulate thephysiological system back into a homeostatic state. Neuromodulation tothe targeted regions of interest may exert a change in physiologicalprocesses to interrupt, decrease, or augment one or more physiologicalpathways in a subject to yield the desired physiological perturbation.

The neuromodulation techniques discussed herein may be used to cause aphysiological perturbation in the neuromodulated subject. Thephysiological perturbation may include local changes in the region ofinterest or tissue to which the energy was applied as well as systemicchanges that are the result of the neuromodulation. In certainembodiments, one or more characteristics of the perturbation may beassessed as part of the present techniques. These changes may includechanges in one or more molecules of interest, changes in physiologicalparameters of the subject, displacement, enlargement, or othermorphological changes to one or more tissue structures of the subject,changes in cell populations in the subject, a change in flow parametersof blood or other fluids, etc. In one embodiment, the physiologicalperturbation causes a change in concentration (e.g., increased,decreased) of a molecule of interest and/or a change in characteristicsof a molecule of interest. That is, the perturbation may includeselective modulation of the tissue production or release of one or moremolecules of interest (e.g., a first molecule of interest, a secondmolecule of interest, and so on) and may refer to modulating orinfluencing a concentration (circulating, tissue) or characteristics(covalent modification) of a molecule as a result of energy applicationto one or more regions of interest (e.g., a first region of interest, asecond region of interest, and so on) in one or more tissues (e.g., afirst tissue, a second tissue, and so on). Modulation of a molecule ofinterest may include changes in characteristics of the molecule such asexpression, secretion, translocation of proteins and direct activitychanges. Modulation may be driven based on the effect of the appliedenergy on ion channel either driving nerve activity and function itselfor modulation of neighboring non-neuronal cells as a result of moleculesderived from the neural activity or direct activation within thenon-neuronal cell. Modulation of a molecule of interest may also referto maintaining a desired concentration of the molecule, such thatexpected changes or fluctuations in concentration do not occur as aresult of the neuromodulation. Modulation of a molecule of interest mayrefer to causing changes in molecule characteristics, such asenzyme-mediated covalent modification (changes in phosphorylation,acetylation, ribosylation, etc.). That is, it should be understood thatselective modulation of a molecule of interest may refer to moleculeconcentration and/or molecule characteristics. The molecule of interestmay be a biological molecule, such as one or more of carbohydrates(monosaccharaides, polysaccharides), lipids, nucleic acids (DNA, RNA),or proteins. In certain embodiments, the molecule of interest may be asignaling molecule such as a hormone (an amine hormone, a peptidehormone, or a steroid hormone).

Certain embodiments described herein provide neuromodulation techniquesfor the diagnosis of metabolic dysfunction. In one embodiment, thediagnosis may be a presence or absence of glucose metabolism dysfunctionand associated disorders. Glucose regulation is complex and involvesdifferent local and systemic metabolic pathways. Application of energyto targeted region/s of interest causes characteristic changes in thesemetabolic pathways that affect glucose regulation. In some embodiments,modulation at one or more regions of interest may be used to identifydisorders including but not limited to, diabetes (i.e., type 1 or type 2diabetes), hyperglycemia, hyperlipidemia, sepsis, trauma, infection,physiological stress, diabetes-associated dementia, obesity, or othereating or metabolic disorders. In one example, physiological stress maybe medically defined to include a variety of acute medical conditions(infection, severe injury/trauma, heart attack, bypass) as well assurgical instances with presentation of hyperglycemia. The targetedperturbation via neuromodulation may induce changes in glucoregulatoryhormones in the blood or tissue to cause a deviation from glucoseconcentration. The targeted perturbation via neuromodulation may alsoinduce changes in activity of sensory or effector neurons within themetabolic physiological control system, and response of this system maybe analyzed by imaging (e.g., MM) or electrically recording (e.g., ECG)or other physiological monitoring. The change to these systems as aresult of neuromodulation may be different in healthy versus diseasedpatients. In a healthy subject, the perturbation may be cleared overtime such that glucose concentration returns to normal. Further,physiological perturbations may be used to identify subjects without adisease diagnosis, but who are pre-diabetic. In one embodiment, thephysiological perturbation is used to identify subjects with insulinresistance, who may or may not be identified as diabetic.

Certain embodiments described herein provide neuromodulation techniquesfor the diagnosis of control of inflammation and immune function andassociated disorders. Regulation of the inflammatory and activity statusof immune cells involves different local and systemic neural, humoral,and cellular pathways. Application of energy to targeted region/s ofinterest causes characteristic changes in these inflammatory andanti-inflammatory pathways that affect circulating cytokine andneurotransmitter concentrations and therefore immune cell activity andstates. In some embodiments, modulation at one or more regions ofinterest may be used to identify disorders including but not limited to,rheumatoid arthritis, irritable bowel disease, psoriasis, or otherchronic inflammatory or related disorders. The targeted perturbation vianeuromodulation may induce changes in immune markers such asinflammatory cytokine, hormones, or neurotransmitters in the blood ortissue to cause activity changes in resident or circulating immunecells. The targeted perturbation via neuromodulation may also inducechanges in activity of sensory or effector neurons within theinflammatory control systems, and response of this system may beanalyzed by imaging or electrically recording. The change to thesesystems as a result of neuromodulation may be different in healthyversus diseased patients. In a healthy patient, the perturbations maycause temporary changes to physiology or changes that are lower inmagnitude, while in diseased patients the perturbations may be longerlasting. Further, physiological perturbations may be used to identifypatients without a current disease diagnosis, or who are pre-symptomaticor developing a chronic inflammatory disease.

To that end, the present techniques relate to targeted modulation ofsynapses at axon terminals in a tissue via a direct application ofenergy by an energy source to cause a change that results in ameasurable physiological perturbation (e.g., a change in a circulatingmolecule concentration or a suite of concentrations changes forming acharacteristic physiological profile). The targeted synapses may includeaxoextracellular synapses formed between presynaptic axon terminals andpostsynaptic non-neuronal cells. In addition, while certain disclosedembodiments are discussed in the context of axoextracellular synapses,it should be understood that the axon terminals may form axosecretory,axosynaptic, axosomatic or axoextracellular synapses, and thatadditionally or alternatively, these synaptic types are contemplated asbeing selectively modulated, as provided herein. Further, certain axonterminals may terminate in interstitial or body fluid that may alsoexperience neurotransmitter release as a result of the modulation. Thedisclosed synapses may be modulated to alter an activity in thesynapses, e.g., a release of neurotransmitters from the presynaptic axonterminals, as a result of the energy application. Accordingly, thealtered activity may lead to local effects and/or non-local (e.g.,systemic) effects to cause the overall profile of physiological changesassociated with the desired or targeted physiological perturbation. Thepresent techniques permit energy to be focused in a targeted manner on avolume of tissue that includes certain axon terminals to preferentiallydirectly activate the targeted axon terminals to achieve desiredphysiological perturbations. In this manner, the targeted axon terminalswithin a region of interest are activated while, in certain embodiments,axon terminals in the same organ or tissue structure but that areoutside of the region of interest are not activated. Because organs andtissue structures may include different types of axon terminals thatform synapses with different types of postsynaptic non-neuronal cells,the region of interest may be selected that includes particular types ofaxon terminals that, when activated, yield the desired targetedphysiological perturbation. Accordingly, the modulation may target aspecific type of axon terminal on the basis of the presynaptic neurontype, the postsynaptic cell type, or both.

For example, in one embodiment, the type of axon terminal may be an axonterminal forming an axoextracellular synapse with a resident (i.e.,tissue-resident or non-circulating) liver, pancreatic, orgastrointestinal tissue cell. That is, the axoextracellular synapse isformed at a junction between an axon terminal and a nonneuronal cell orinterstitial or body fluid. Accordingly, the application of energy leadsto modulation of function in the region of interest. However, it shouldbe understood that, based on the population of axon terminal types andthe characteristics of the presynaptic neuron type and postsynapticcells (e.g., immune cells, lymph cells, mucosal cells, muscle cells,etc.) of the axoextracellular synapse, different targeted physiologicaleffects may be achieved. Further, as noted, the axon terminals mayterminate in interstitial or body fluid that may also experienceneurotransmitter release as a result of the modulation. Accordingly,applying energy to a region of interest in a tissue of a subject mayactivate axon terminals (and, where applicable, their associatedaxoextracellular synapse) within the region of interest while untargetedaxon terminals (and associated synapses) outside of the region ofinterest may be unaffected. However, because modulation may result insystemic effects, untargeted axon terminals outside of the region ofinterest may experience certain systemic changes as a result of theactivation of the axon terminals within the region of interest. Asprovided herein, preferential activation or direct activation may referto the cells or structures (e.g., synapses) that experience directapplication of energy (e.g., the energy is applied directly to the cellsor structures) and are within a region of interest. For example, axonterminals, axoextracellular synapses, and/or postsynaptic non-neuronalcells or interstitial or body fluid within the region of interest maydirectly experience the applied energy as provided herein. Preferentialor direct activation may be considered in contrast to areas outside of aregion of interest that do not experience direct energy application,even if such areas nonetheless undergo physiological changes as a resultof the energy application.

Neuromodulation is a technique in which energy from an external energysource is applied to certain areas of the nervous system to activate orincrease the nerve or nerve function and/or block or decrease the nerveor nerve function. In certain neuromodulation techniques, one or moreelectrodes are applied at or near target nerves, and the application ofenergy is carried through the nerve (e.g., as an action potential) tocause a physiological response in areas of the downstream of the energyapplication site. However, because the nervous system is complex, it isdifficult to predict the scope and eventual endpoint of thephysiological response for a given energy application site. In oneexample, stimulation of axon terminals releasesneurotransmitter/neuropeptide or induces altered neurotransmitterrelease in a vicinity of neighboring non-neuronal cells such assecretory or other cells and modulates cell activity of the neighboringor nearby non-neuronal cells, including the postsynaptic cells.

Benefits of the present techniques include local modulation at theregion of interest of the tissue to achieve physiological perturbationsthat may be used to assess a subject condition. Further, the localmodulation may involve direct activation of a relatively small region oftissue (e.g., less than 25% of a total tissue volume) to achieve theseeffects. In this manner, the total applied energy is relatively small toachieve a desired physiological perturbation. In certain embodiments,the applied energy may be from a non-invasive extracorporeal energysource (e.g., ultrasound energy source, mechanical vibrator). Forexample, a focused energy probe may apply energy through a subject'sskin and is focused on a region of interest of an internal tissue. Suchembodiments achieve the desired physiological perturbation withoutinvasive procedures or without side effects that may be associated withother types of procedures or therapy.

In certain embodiments, techniques for neuromodulation are provided inwhich energy from an energy source (e.g., an external or extracorporealenergy source) is applied to axon terminals in a manner such that theinduced physiological perturbation, for example, neurotransmitterrelease, at the site of focus of the energy application, e.g., the axonterminals, is triggered in response to the energy application and not inresponse to an action potential. That is, the application of energydirectly to the axon terminals acts in lieu of an action potential tofacilitate neurotransmitter release into a neuronal junction (i.e.,synapse) with a non-neuronal cell. The application of energy directly tothe axon terminals further induces an altered neurotransmitter releasefrom the axon terminal within the synapse (e.g., axoextracellularsynapse) into the vicinity of neighboring non-neuronal cells. In oneembodiment, the energy source is an extracorporeal energy source, suchas an ultrasound energy source or a mechanical vibrator. In this manner,non-invasive and targeted neuromodulation may be achieved directly atthe site of energy focus rather than via stimulation at an upstream sitethat in turn triggers an action potential to propagate to the downstreamsite target and to activate downstream targets.

In certain embodiments, the target tissues are internal tissues ororgans that are difficult to access using electrical stimulationtechniques with electrodes. Contemplated tissue targets includegastrointestinal (GI) tissue (stomach, intestines), muscle tissue(cardiac, smooth and skeletal), epithelial tissue (epidermal, organ/GIlining), connective tissue, glandular tissues (exocrine/endocrine),organ tissue, etc. In one example, focused application of energy at aneuromuscular junction facilitates neurotransmitter release at theneuromuscular junction without an upstream action potential. In oneembodiment, contemplated targets or regions of interest for modulationmay include portions of a pancreas responsible for controlling insulinrelease or portions of the liver responsible for glucose regulation. Inanother embodiment, contemplated regions of interest may be located inthe liver. In another embodiment, contemplated regions of interest maybe located in the spleen. However, it should be understood that theseembodiments are by way of example.

To that end, the disclosed neuromodulation techniques may be used inconjunction with a neuromodulation system. FIG. 1 is a schematicrepresentation of a system 10 for neuromodulation to achieveneurotransmitter release and/or activate components (e.g., thepresynaptic cell, the postsynaptic cell) of a synapse in response to anapplication of energy. The depicted system includes a pulse generator 14coupled to an energy application device 12 (e.g., an ultrasoundtransducer). The energy application device 12 is configured to receiveenergy pulses, e.g., via leads or wireless connection, that in use aredirected to a region of interest of an internal tissue or an organ of asubject, which in turn results in a targeted physiological perturbation.In certain embodiments, the pulse generator 14 and/or the energyapplication device 12 may be implanted at a biocompatible site (e.g.,the abdomen), and the lead or leads couple the energy application device12 and the pulse generator 14 internally. For example, the energyapplication device 12 may be a MEMS transducer, such as a capacitivemicromachined ultrasound transducer.

In certain embodiments, the energy application device 12 and/or thepulse generator 14 may communicate wirelessly, for example with acontroller 16 that may in turn provide instructions to the pulsegenerator 14. In other embodiments, the pulse generator 14 may be anextracorporeal device, e.g., may operate to apply energy transdermallyor in a noninvasive manner from a position outside of a subject's body,and may, in certain embodiments, be integrated within the controller 16.In embodiments in which the pulse generator 14 is extracorporeal, theenergy application device 12 may be operated by a caregiver andpositioned at a spot on or above a subject's skin such that the energypulses are delivered transdermally to a desired internal tissue. Oncepositioned to apply energy pulses to the desired site, the system 10 mayinitiate neuromodulation to achieve targeted physiological perturbationor clinical effects.

In certain embodiments, the system 10 may include an assessment device20 that is coupled to the controller 16 and that assessescharacteristics that are indicative of whether the targetedphysiological perturbation of the modulation have been achieved. In oneembodiment, the targeted physiological perturbation may be local. Forexample, the modulation may result in local tissue or function changes,such as tissue structure changes, local change of concentration ofcertain molecules, tissue displacement, increased fluid movement, etc.

The modulation may result in systemic or non-local changes, and thetargeted physiological perturbation may be related to a change inconcentration of circulating molecules or a change in a characteristicof a tissue that does not include the region of interest to which energywas directly applied. In one example, tissue displacement may be a proxymeasurement for a desired modulation, and displacement measurementsbelow an expected displacement value may result in modification ofmodulation parameters until an expected displacement value is induced.Accordingly, the assessment device 20 may be configured to assessmolecule concentration or activity changes in some embodiments. Forexample, the assessment device 20 may include a chemical sensor for themolecule of interest. In some embodiments, the assessment device 20 maybe an imaging device configured to assess changes in organ size and/orposition, fluid flow changes, RNA or protein expression changes, orother indicators of physiological perturbations. In another embodiment,the assessment device 20 may be a circulating glucose monitor and/or acontinuous glucose monitor that measures interstitial fluid glucose. Theassessment device 20 may be an MM device or other devices than iscapable of acquiring image data of a subject to identify systemicchanges. The assessment device 20 may be an ECG or other physiologicalmonitor. While the depicted elements of the system 10 are shownseparately, it should be understood that some or all of the elements maybe combined with one another. Further, some or all of the elements maycommunicate in a wired or wireless manner with one another.

Based on the assessment, the modulation parameters of the controller 16may be altered. For example, if a desired modulation is associated witha change in concentration (circulating concentration or tissueconcentration of one or more molecules) within a defined time window(e.g., 5 minutes, 30 minutes after a procedure of energy applicationstarts) or relative to a baseline at the start of a procedure, a changeof the modulation parameters such as pulse frequency or other parametersmay be desired, which in turn may be provided to the controller 16,either by an operator or via an automatic feedback loop, for defining oradjusting the energy application parameters or modulation parameters ofthe pulse generator 14.

The system 10 as provided herein may provide energy pulses according tovarious modulation parameters. For example, the modulation parametersmay include various stimulation time patterns, ranging from continuousto intermittent. With intermittent stimulation, energy is delivered fora period of time at a certain frequency during a signal-on time. Thesignal-on time is followed by a period of time with no energy delivery,referred to as signal-off time. The modulation parameters may alsoinclude frequency and duration of a stimulation application. Theapplication frequency may be continuous or delivered at various timeperiods, for example, within a day or week. In one example, themodulation may be specified relative to a time of day (morning, evening,night) or relative to meals (fasted, nonfasted) The treatment durationto cause the physiological perturbations may last for various timeperiods, including, but not limited to, from a few minutes to severalhours. In certain embodiments, treatment duration with a specifiedstimulation pattern may last for one hour, repeated at, e.g., 72 hourintervals. In certain embodiments, energy may be delivered at a higherfrequency, say every three hours, for shorter durations, for example, 30minutes. The application of energy, in accordance with modulationparameters, such as the treatment duration and frequency, may beadjustably controlled to achieve a desired result.

FIG. 2 is a block diagram of certain components of the system 10. Asprovided herein, the system 10 for neuromodulation may include a pulsegenerator 14 that is adapted to generate a plurality of energy pulsesfor application to a tissue of a subject. The pulse generator 14 may beseparate or may be integrated into an external device, such as acontroller 16. The controller 16 includes a processor 30 for controllingthe device. Software code or instructions are stored in memory 32 of thecontroller 16 for execution by the processor 30 to control the variouscomponents of the device. The controller 16 and/or the pulse generator14 may be connected to the energy application device 12 via one or moreleads 33 or wirelessly.

The controller 16 also includes a user interface with input/outputcircuitry 34 and a display 36 that are adapted to allow a clinician toprovide selection inputs or modulation parameters to modulationprograms. Each modulation program may include one or more sets ofmodulation parameters including pulse amplitude, pulse width, pulsefrequency, etc. The pulse generator 14 modifies its internal parametersin response to the control signals from controller device 16 to vary thestimulation characteristics of energy pulses transmitted through lead 33to an subject to which the energy application device 12 is applied. Anysuitable type of pulse generating circuitry may be employed, includingbut not limited to, constant current, constant voltage,multiple-independent current or voltage sources, etc. The energy appliedis a function of the current amplitude and pulse width duration. Thecontroller 16 permits adjustably controlling the energy by changing themodulation parameters and/or initiating energy application at certaintimes or cancelling/suppressing energy application at certain times. Inone embodiment, the adjustable control of the energy application deviceis based on information about a concentration of one or more moleculesin the subject (e.g., a circulating molecule). If the information isfrom the assessment device 20, a feedback loop may drive the adjustablecontrol. For example, a diagnosis may be made based on circulatingglucose concentration, as measured by the assessment device 20, inresponse neuromodulation. When the concentration is above apredetermined threshold or range, the controller 16 may initiate atreatment protocol of energy application to a region of interest (e.g.,liver) and with modulation parameters that are associated with areduction in circulating glucose. The treatment protocol may usedifferent modulation parameters than those used in the diagnosisprotocol (e.g., higher energy levels, more frequent application). Thecontroller may initiate a treatment protocol of energy application to aregion of interest (e.g., spleen or immune tissue) and with modulationparameters that are associated with an increase or reduction incirculating cytokines or immune cell number, activity, or phenotype.

In one embodiment, the memory 32 stores different operating modes thatare selectable by the operator. For example, the stored operating modesmay include instructions for executing a set of modulation parametersassociated with a particular treatment site, such as regions of interestin the liver, pancreas, gastrointestinal tract, spleen. Different sitesmay have different associated modulation parameters. Rather than havingthe operator manually input the modes, the controller 16 may beconfigured to execute the appropriate instruction based on theselection. In another embodiment, the memory 32 stores operating modesfor different types of procedures. For example, activation may beassociated with a different stimulating pressure or frequency rangerelative to those associated with depressing or blocking tissuefunction. In a specific example, when the energy application device isan ultrasound transducer, the time-averaged power (temporal averageintensity) and peak positive pressure are in the range of 1mW/cm2-30,000 mW/cm2 (temporal average intensity) and 0.1 MPa to 7 MPa(peak pressure). In one example, the temporal average intensity is lessthan 35 W/cm2 in the region of interest to avoid levels associated withthermal damage & ablation/cavitation. In another specific example, whenthe energy application device is a mechanical actuator, the amplitude ofvibration is in the range of 0.1 to 10 mm. The selected frequencies maydepend on the mode of energy application, e.g., ultrasound or mechanicalactuator. The controller 16 may be capable of operating in a diagnosisprotocol mode and the results of the diagnosis may trigger a change to atreatment operating mode. For example, a diagnosis mode may applyneuromodulating energy once or repeatedly within a relatively short timewindow (1 hour), while a treatment operating mode may involve repeatedneuromodulation events (e.g., daily, hourly) during a treatmentprotocol.

The system may also include an imaging device that facilitates focusingthe energy application device 12. In one embodiment, the imaging devicemay be integrated with or the same device as the energy applicationdevice 12 such that different ultrasound parameters (frequency,aperture, or energy) are applied for selecting (e.g., spatiallyselecting) a region of interest and for focusing energy to the selectedregion of interest for targeting and subsequently neuromodulation. Inanother embodiment, the memory 32 stores one or more targeting orfocusing modes that is used to spatially select the region of interestwithin an organ or tissue structure. Spatial selection may includeselecting a subregion of an organ to identify a volume of the organ thatcorresponds to a region of interest. Spatial selection may rely on imagedata as provided herein. Based on the spatial selection, the energyapplication device 12 may be focused on the selected volumecorresponding to the region of interest. For example, the energyapplication device 12 may be configured to first operate in thetargeting mode to apply a targeting mode energy that is used to captureimage data to be used for identifying the region of interest. Thetargeting mode energy is not at levels and/or applied with modulationparameters suitable for preferential activation. However, once theregion of interest is identified, the controller 16 may then operate ina treatment mode according to the modulation parameters associated withpreferential activation.

The controller 16 may also be configured to receive inputs related tothe targeted physiological perturbations as an input to the selection ofthe modulation parameters. For example, when an imaging modality is usedto assess a tissue characteristic, the controller 16 may be configuredto receive a calculated index or parameter of the characteristic. Basedon whether the index or parameter is above or below a predefinedthreshold, a diagnosis may be made, and an indication of the diagnosismay be provided (e.g., via a display). In one embodiment, the parametercan be a measure of tissue displacement of the affected tissue or ameasure of depth of the affected tissue. Other parameters may includeassessing a concentration of one or more molecules of interest (e.g.,assessing one or more of a change in concentration relative to athreshold or a baseline/control, a rate of change, determining whetherconcentration is within a desired range). Further, the energyapplication device 12 (e.g., an ultrasound transducer) may operate undercontrol of the controller 16 to a) acquire image data of a tissue thatmay be used to spatially select a region of interest within the targettissue b) apply the modulating energy to the region of interest and c)acquire image to determine that the targeted physiological perturbationhas occurred (e.g., via displacement measurement). In such anembodiment, the imaging device, the assessment device 20 and the energyapplication device 12 may be the same device.

FIG. 3 is a specific example in which the energy application device 12includes an ultrasound transducer 42 that is capable of applying energyto a target tissue 43, e.g., a liver, a spleen, a pancreas. The energyapplication device 12 may include control circuitry for controlling theultrasound transducer 42. The control circuitry of the processor 30(FIG. 2) may be integral to the energy application device 12 (e.g., viaan integrated controller 16) or may be a separate component. Theultrasound transducer 42 may also be configured to acquire image data toassist with spatially selecting a desired or targeted region of interestand focusing the applied energy on the region of interest of the targettissue or structure.

The desired target tissue 43 may be an internal tissue or an organ thatincludes synapses of axon terminals and non-neuronal cells. The synapsesmay be stimulated by direct application of energy to the axon terminalswithin a field of focus of the ultrasound transducer 42 focused on aregion of interest 44 of the target tissue 43 to cause release ofmolecules into the synaptic space, e.g., the release ofneurotransmitters and/or the change in ion channel activity in turncauses downstream effects. Sensory synapses (or communicating cells) mayalso be stimulated by direct application of energy to peripheralterminals and neurons within a field of focus of the ultrasoundtransducer focused on a region of interest of the target tissue andcause direct neural signaling back to the CNS (i.e., driving ormimicking sensory inputs to CNS control centers, ganglia, and nuclei).The region of interest may be selected to include a certain type of axonterminal, such as an axon terminal of a particular neuron type and/orone that forms a synapse with a certain type of non-neuronal cell.Accordingly, the region of interest 44 may be selected to correspond toa portion of the target tissue 43 with the desired axon terminals (andassociated non-neuronal cells). The energy application may be selectedto preferentially trigger a release of one or more molecules such asneurotransmitters from the nerve within the synapse or directly activatethe non-neuronal cell itself through direct energy transduction (i.e.,mechanotransduction or voltage-activated proteins within thenon-neuronal cells), or cause an activation within both the neural andnon-neuronal cells that elicits a desired physiological effect. Theregion of interest may be selected as the site of nerve entry into theorgan. In one embodiment, liver stimulation or modulation may refer to amodulation of the region of interest 44 at or adjacent to the portahepatis.

The energy may be focused or substantially concentrated on a region ofinterest 44 and to only part of the internal tissue 43, e.g., less thanabout 50%, 25%, 10%, or 5% of the total volume of the tissue 43. In oneembodiment, energy may be applied to two or more regions of interest 44in the target tissue 43, and the total volume of the two or more regionsof interest 44 may be less than about 90%, 50%, 25%, 10%, or 5% of thetotal volume of the tissue 43. In one embodiment, the energy is appliedto only about 1%-50% of the total volume of the tissue 43, to only about1%-25% of the total volume of the tissue 43, to only about 1%-10% of thetotal volume of the tissue 43, or to only about 1%-5% of the totalvolume of the tissue 43. In certain embodiments, only axon terminals inthe region of interest 44 of the target tissue 43 would directly receivethe applied energy and release neurotransmitters while the unstimulatedaxon terminals outside of the region of interest 44 do not receivesubstantial energy and, therefore, are not activated/stimulated in thesame manner. In some embodiments, axon terminals in the portions of thetissue directly receiving the energy would induce an alteredneurotransmitter release. In this manner, tissue subregions may betargeted for neuromodulation in a granular manner, e.g., one or moresubregions may be selected. In some embodiments, the energy applicationparameters may be chosen to induce preferential activation of eitherneural or non-neuronal components within the tissue directly receivingenergy to induce a desired combined physiological effect. In certainembodiments, the energy may be focused or concentrated within a volumeof less than about 25 mm³. In certain embodiments, the energy may befocused or concentrated within a volume of about 0.5 mm³-50 mm³. A focalvolume and a focal depth for focusing or concentrating the energy withinthe region of interest 44 may be influenced by the size/configuration ofthe energy application device 12. The focal volume of the energyapplication may be defined by the field of focus of the energyapplication device 12.

As provided herein, the energy may be substantially applied only to theregion or regions of interest 44 to preferentially activate the synapsein a targeted manner to achieve targeted physiological perturbations andis not substantially applied in a general or a nonspecific manner acrossthe entire tissue 43. Accordingly, only a subset of a plurality ofdifferent types of axon terminals in the tissue 43 is exposed to thedirect energy application. The disclosed techniques may be used inassessment of subject condition as a result of the perturbations causedby neuromodulation. The disclosed techniques may use direct assessmentsof tissue condition or function as the targeted physiologicalperturbations. The techniques may also be used to detect deviation fromor toward a homeostatic state or set-point of a physiologicalneuroimmune, neurohormonal, and/or nerve reflex system. The assessmentmay occur before (i.e., baseline assessment), during, and/or after theneuromodulation. The assessment techniques may include at least one offunctional magnetic resonance imaging, diffusion tensor magneticresonance imaging, positive emission tomography, acoustic monitoring,thermal monitoring, or chemical sensing (e.g., immunochemical sensing).The assessment techniques may also include protein and/or moleculeconcentration assessment. The images from the assessment techniques maybe received by the system for automatic or manual assessment. Based onthe image data, the modulation parameters may also be modified. Forexample, a change in organ size or displacement may be utilized as amarker of local neurotransmitter concentration, and used as a surrogatemarker for exposure of local cells to phenotype modulatingneurotransmitters, and effectively as a marker of predicted effect onglucose metabolic pathways. The local concentration may refer to aconcentration within a field of focus of the energy application.

Additionally or alternatively, the system may assess the presence orconcentration of one or more molecules in the tissue or circulating inthe blood. The concentration in the tissue may be referred to as a localconcentration or resident concentration. Tissue may be acquired by afine needle aspirate, and the assessment of the presence or levels ofmolecules of interest (e.g., metabolic molecules, markers of metabolicpathways, peptide transmitters, catecholamines) may be performed by anysuitable technique known to one of ordinary skilled in the art.

In other embodiments, the targeted physiological perturbations mayinclude, but are not limited to, tissue displacement, tissue sizechanges, a change in concentration of one or more molecules (eitherlocal, non-local, or circulating concentration), a change in gene ormarker expression, afferent activity, and cell migration, etc. Forexample, tissue displacement (e.g., liver displacement) may occur as aresult of energy application to the tissue. By assessing the tissuedisplacement (e.g., via imaging), other effects may be estimated. Forexample, a certain displacement may be characteristic of a particularchange in molecule concentration. In one example, a 5% liverdisplacement may be indicative of or associated with a desired reductionin circulating glucose concentration based on empirical data. In anotherexample, the tissue displacement may be assessed by comparing referenceimage data (tissue image before application of energy to the tissue) topost-treatment image data (tissue image taken after application ofenergy to the tissue) to determine a parameter of displacement. Theparameter may be a maximum or average displacement value of the tissue.If the parameter of displacement is greater than a thresholddisplacement, the application of energy may be assessed as being likelyto have caused the desired targeted physiological perturbation.

In one example, the present techniques may be used to diagnose a subjectwith a metabolic disorder. The present techniques may also be used todiagnose and/or treat subjects with disorders of glucose regulation.Accordingly, the present techniques may be used to promote homeostasisof a molecule of interest or to promote a desired circulatingconcentration or concentration range of one or more molecules ofinterest (e.g., glucose, insulin, glucagon, or a combination thereof).In one embodiment, the present techniques are used to assess clinicalconditions associated with circulating (i.e., blood) glucose levels.

In one embodiment, thresholds of glucose concentration may be used toidentify blood glucose levels outside of or in the normal range as partof diagnosis of metabolic dysfunction.

Fasted:

Less than 50 mg/dL (2.8 mmol/L): Insulin Shock50-70 mg/dL (2.8-3.9 mmol/L): low blood sugar/hypoglycemia70-110 mg/dL (3.9-6.1 mmol/L): normal110-125 mg/dL (6.1-6.9 mmol/L): elevated/impaired (pre-diabetic)125 (7 mmol/L): diabeticNon-fasted (postprandial approximately 2 hours after meal):70-140 mg/dL: Normal140-199 mg/dL (8-11 mmol/L): Elevated or “borderline”/prediabetesMore than 200 mg/dL: (11 mmol/L): Diabetes

However, as provided herein, the present techniques permit assessment ofperturbations as a result of neuromodulation, even in the absence offasting or nonfasting glucose information. In this manner, patientnoncompliance or misreporting may be eliminated as a confounding factorto assessment of metabolic dysfunction. That is, a subject with anunknown fasting or nonfasting state may be perturbed via neuromodulationinto an approximated or pseudofasting state, even if that subject is notfasted, e.g., has recently eaten. As provided herein, an approximatedfasting state may refer to a state of a patient after (e.g., within 1-6hours after, within 30 minutes after) neuromodulating energy is appliedto a region of interest, e.g., liver, pancreas, GI, associated withmetabolism. Based on the subject's profile or response toneuromodulation, the absence or presence of metabolic dysfunction may beidentified.

For example, the above normal glucose ranges may be used as recoverytargets to track a time to recovery after changes in glucose levelsfollowing a perturbation caused by neuromodulation. In another example,a subject with an elevated fasted glucose level that does not experiencea change in glucose levels after neuromodulation is diagnosed with adeficit in insulin production (e.g., a pancreatic disorder). Incontrast, if that same subject drops glucose levels significantly (morethan an expected amount) after neuromodulation that may indicate aproblem in the brain control center (e.g., glucose setpoint).Accordingly, in one embodiment, the response to perturbation permitscategorizing subjects based on their particular response profile (e.g.,a change in glucose vs. a lack of change in glucose concentration). Inone embodiment, the perturbation is induced and the concentration of oneor more molecules of interest is assessed. Based on the concentrationbeing above or below a threshold, the subject is categorized in a firstcategory or a second category. Further, their response profile may becategorized relative to their baseline state. A subject with normalglucose levels that experiences no change in glucose followingneuromodulation-induced perturbation is categorized differently (e.g.,placed in a first category) than a subject with elevated glucose levelsthat experiences no change in glucose following neuromodulation-inducedperturbation (e.g., placed in a second category). Accordingly, a levelof change relative to baseline is used to categorize subjects. A changerelative to baseline that is less than a predetermined threshold isassociated with a first category while a change that is greater than thepredetermined threshold is associated with a second category. It shouldbe understood that the disclosed categories may include additionalsubcategories for a subject.

In another example, the present techniques cause perturbations in bothfasted and non-fasted subjects. Accordingly, subjects may prefer to besubject to diagnostic neuromodulation that does not involve dietarychanges. In addition, in certain embodiments, the subjects also need notbe subject to intravenous administration of pharmacologic agents (e.g.,glucose, insulin).

In another example, as provided herein, neuromodulation of lymphatictissue may result in perturbation of immune activity or function. Incertain embodiments, neuromodulation of a lymph node results in localenlargement of the lymph node relative to a contralateral lymph node.The enlargement, or hypertrophy, may be associated with a change inperi-lymphatic vessel muscle cell tone, longer term recruitment andre-organization of lymphatic vessels around the lymph node, and/ormolecular changes at key barrier tissues (such as high endothelialvenules (HEV) within the lymph node) including alteration of importanttransport proteins (such as aquaporin (water/liquid transport), orCCL21/CXCL13 secretion (cell chemokines)). The perturbations may resultin dramatic shifts in cell densities, cell counts, and the overall lymphnode tissue environment, including the enlargement. Further, activationof the lymph node may result in an activation chain that expands oramplifies the local activation to systemically activate the lymphaticsystem. That is, local stimulation may result in downstream and upstreamactivation of lymphatic systems. Accordingly, local perturbations may beused to activate a systemic immune response. Additional perturbationsmay include alteration of lymphatic fluid flow, immune cell traffickinginto/out of the lymphatic tissue, alteration of immune cell phenotype orlocal immune response, and/or antigen trafficking into/out of thelymphatic tissue. Local stimulation may enable tissue or locationspecific increase in lymphatic fluid or immune cell recruitment.Accordingly, in one embodiment, neuromodulation of lymph tissue and anassessment of perturbations to immune activity (or a lack thereof) maybe used to diagnose immune dysfunction or immune disorders.

FIG. 4 is a flow diagram of a method 50 of causing perturbations vianeuromodulation that may be used as part of a diagnosis protocol. In themethod 50, the region of interest is spatially selected 52. The energyapplication device is positioned such that the energy pulses are focusedat the desired region of interest at step 54, and the pulse generatorapplies a plurality of energy pulses to the region of interest of thetarget tissue at step 56 to preferentially activate a subset of synapsesin the target tissue, e.g., to stimulate the axon terminal to releaseneurotransmitters and/or induce altered neurotransmitter release and/orinduce altered activity in the non-neuronal cell (within the synapse) tocause a targeted physiological perturbation at step 58 as providedherein. In certain embodiments, the method may include a step ofassessing the effect of the perturbation. For example, one or moredirect or indirect assessments of a state of tissue function orcondition may be used.

In one embodiment, assessment is performed before and after applyingenergy pulses to assess a change in glucose concentration as a result ofthe modulation. Further, the assessed characteristic or condition may bea value or an index, for example, a flow rate, a concentration, a cellpopulation, or any combination thereof, which in turn may be analyzed bya suitable technique. For example, a relative change exceeding athreshold is used to diagnose a subject. The diagnosis is assessed via ameasured perturbation, such as a presence or absence of an increase intissue structure size (e.g., lymph node size) or a change inconcentration of one or more released molecules (e.g., relative to thebaseline concentration before the neuromodulation). In one embodiment,the perturbation involves an increase in concentration above athreshold, e.g., above a about 50%, 100%, 200%, 400%, 1000% increase inconcentration relative to baseline. The assessment involves tracking adecrease in concentration of a molecule over time, e.g., at least a 10%,20%, 30%, 50%, or 75% decrease in the molecule of interest. Further, forcertain subjects, the diagnosis may be associated with a relativelysteady concentration of a particular molecule in the context of otherclinical events that may tend to increase the concentration of themolecule. That is, a normal subject may respond to glucoseadministration concurrently with or after neuromodulation in a mannerthat is distinguishable from an insulin-resistant subject. The increaseor decrease or other induced and measurable effect is measured within acertain time window from the start of a treatment, e.g., within about 5minutes, within about 30 minutes.

FIG. 5 is a flow diagram of a method 60 for assessing perturbations ofone or more molecules of interest relative to baseline to diagnose asubject or a part of a diagnosis protocol. For example, the disclosedtechniques may be used to categorize subjects as being in a first,second, third, etc., category based on the level of the perturbationresponse and/or the recovery back towards baseline. The baselineconcentration (block 62) may be determined based on the moleculeconcentration at or before the perturbation and may be a single baselineconcentration or an average over several time points before theperturbation. The diagnosis protocol may also include the step ofcausing physiological perturbations (e.g., as in the method 50 of FIG.4) via neuromodulation of a region of interest of a target tissue of thesubject (block 64). The target tissue may be selected based on thediagnosis protocol. For example, diagnosis of glucose metabolismdisorders may involve liver and/or pancreas neuromodulation whilediagnosis of immune dysfunction may involve spleen and/or lymph tissueneuromodulation. The region of interest is selected based on the targettissue as disclosed with regard to FIG. 4. Deviation of theconcentration of one or more molecules of interest relative to baselineis determined (block 66) at one or more time points. Based on assessmentat a time point or a plurality of time points after the application ofenergy to the region of interest, the subject's clinical condition maybe determined (block 68). The time point or time points at which thechange in may be determined based on empirical evidence. For example,certain subjects may see changes in the concentration of the molecule ofinterest by 1, 5, 10, or 60 minutes after neuromodulation. Further, suchchanges may dissipate towards recovery after a certain period of time(e.g., after 3 hours, 12 hours, 24 hours). Accordingly, the assessmentof the change occurs during the time period in which the physiologicalperturbation is expected to be observed in at least a certain populationof subjects or for a certain diagnosis. It should be understood that, inaddition, the concentration at additional time points within therecovery period may be assessed. Further, the concentration of themolecule of interest over a period of time may be assessed for predictedincreases or decreases (or lack of change) associated with one or moreclinical conditions.

The method 60 may be used to diagnose a glucose metabolism disorder. Inone embodiment, the present techniques may cause a physiologicalperturbation that results in an overall decrease in circulating glucoseafter neuromodulation of the liver (e.g., at a region of interest at ornear a porta hepatis), or a change in the concentration or rate ofchange of glucose relative to circulating hormone concentrations (suchas insulin or glucagon). Such changes may be related to the clinicalcondition of the subject. For example, in diabetic subjects orpre-diabetic subjects, the baseline circulating glucose levels may be atlevels greater than 140 mg/dL. After perturbation caused by liverneuromodulation, the perturbed levels of glucose may drop to levels thatare consistent with a nondiabetic subject, e.g., levels below 140 mg/dL.Accordingly, a high baseline circulating glucose and a perturbeddeviation from the baseline caused by neuromodulation of the liver maybe diagnostic of a diabetic or prediabetic subject. In contrast, anormal baseline circulating glucose and no perturbation or a smallperturbation of the circulating glucose caused by neuromodulation of theliver may be diagnostic of a healthy or nondiabetic subject. Further, ahigh baseline circulating glucose and no or a small perturbed deviationfrom the baseline caused by neuromodulation of the liver may bediagnostic of organ dysfunction, with or without the presence ofdiabetes. Accordingly, such subjects are considered to be nonresponsiveto neuromodulation, and alternate therapy is recommended.

The changes in circulating glucose may be assessed in the context ofadditional molecules of interest. For example, the relationship betweenglucose and insulin and resultant perturbations may be indicative of theclinical condition of the subject. In one embodiment, physiologicalperturbation via targeted liver ultrasound causes observable changes incirculating glucose, insulin, cortisol and triglycerides relative tobaseline in diabetic or prediabetic subjects. Perturbations that cause adecrease in glucose without a concurrent change in insulin may beindicative of a first type of glucose metabolism disorder whileperturbations that cause a decrease in glucose with a concurrent changein insulin may be indicative of a second type of glucose metabolismdisorder. That is, the magnitude of the delta, which may be an observedglucose and/or insulin concentration change (or lack thereof), over timemay be a marker of a glucose metabolism disorder. In one example,insulin resistance is characterized by a subject with elevatedcirculating insulin. In response to neuromodulation of the pancreasaccording to the present techniques, there is an initial increase incirculating insulin as the mechanical wave likely causes the release ofpancreatic insulin stores. However, subsequent to the release ofexisting pancreatic insulin stores that cause the initial increase,there is a subsequent overall decrease in the circulating insulinrelative to both the levels seen in the initial increase as well asrelative to the initial or baseline circulating insulin beforepancreatic neuromodulation. Observing a response to pancreaticneuromodulation in a subject with elevated circulating insulin and/orelevated circulating glucose above a threshold concurrently with orseparate from liver neuromodulation and that tracks the initial increasefollowed by a decrease in circulating insulin may be used to categorizea subject as insulin resistant without requiring that the subject befasted. Further, a lack of such a characteristic response may be used toidentify subjects that exhibit elevated circulating insulin and/orelevated circulating glucose above a threshold but that are not insulinresistant and that may exhibit such concentrations as a result of otherdysfunctional metabolic pathways. Through observing one or more types ofperturbations caused by neuromodulation, subjects may be codified orgrouped into different categories based on the particular profile ofchanges. Follow-up diagnosis protocols may track deviation from previouscategorization and may, in turn, be used to track subject prognosis.

Lymphatic tissue neuromodulation may be used to perturb populations ofimmune cells produced by lymphatic structures. In one embodiment,neuromodulation of the lymph nodes may result in an increase in thepopulation of lymphocytes circulating in the lymphatic fluid.Accordingly, the local concentration profile of type 1(pro-inflammatory) cytokines (e.g., IL-12, TNF-alpha, IFN-gamma, IL-2,TNF-beta) and type 2 (anti-inflammatory) cytokines (e.g., IL-4, IL-10,IL-13, IL-6) may be assessed. In one embodiment, neuromodulation of thelymph nodes may result in an increase or decrease in B or T cellscirculating on the lymphatic fluid, or an increase or decrease in B or Tcells or dendritic cells recruited into lymphatic tissue. Accordingly,neuromodulation of lymphatic tissue may result in a chance in cellmigration patterns. Such migration patterns may be observed using invivo bioluminescence imaging. Other characteristics may include a changein lymph drainage patterns. In certain embodiments, these changes may becharacteristics of the perturbation used to diagnose a subject. Healthysubjects, immunocompromised or immunodeficient subjects, and subjectswith a hyperactive immune response may experience differentperturbations as a result of neuromodulation of lymph tissue.Characteristics of these perturbations may be assessed to createprofiles. A subject with an unknown clinical condition may be diagnosedaccording to a closest match to a characteristic profile. For example,an immunodeficient subject may have a characteristic lack of response ora low level of perturbations in response to targeted splenicneuromodulation. In certain embodiments, the occipital, auricular,cervical, axillary, inguinal, pulmonary, mediastinal, intraabdominal, orepitrochlear lymph nodes may be targeted.

FIG. 6 is a method 69 of identifying individual portions of a disorderedmetabolic pathway based on perturbation patterns. Energy may be appliedto individual regions of interest (e.g., a first region of interest, asecond region of interest) in different organs (e.g., a first organ, asecond organ) to induce neuromodulation and resultant perturbations(block 70). The energy application may be in series (e.g., at differenttimes) to permit assessment of the characteristics of the perturbationat each organ (block 71). The characteristic of the perturbation includemeasurable physiological changes that are assessed. Based on theassessment, a clinical condition may be diagnosed (block 72). Forexample, energy may be applied to a liver and a pancreas at differenttime points to identify point of dysfunction in glucose metabolism. Asubject with a diseased pancreas may respond differently than a subjectwho is insulin resistant. That is, an insulin resistant subject mayrespond with increased levels of circulating insulin as a result oftargeted pancreatic stimulation. However, a subject with a diseasedpancreas may be incapable of generating insulin and, therefore, may notexhibit the characteristic of increased circulating insulin as a resultof neuromodulation of the pancreas. An insulin-resistant patient mayalso respond with a decrease in blood glucose that does not coincidewith an increase in insulin concentration after stimulation ofperipheral sensory sites (such as the liver or GI tract), as stimulationof these sites acts on other CNS based sensory sites that are deficit inglucose sensor and control. Other techniques may not distinguish betweenthese scenarios.

The energy application device 12 may be configured as an extracorporealnon-invasive device or an internal device, e.g., a minimally invasivedevice. As noted, the energy application device 12 may be anextracorporeal noninvasive ultrasound transducer or mechanical actuator.For example, FIG. 7 shows an embodiment of the energy application device12 configured as a handheld ultrasound probe including an ultrasoundtransducer 74. However, it should be understood that other noninvasiveimplementations are also contemplated, including other methods toconfigure, adhere, or place ultrasound transducer probes over ananatomical target. Further, in addition to handheld configurations, theenergy application device 12 may include steering mechanisms responsiveto instructions from the controller 16. The steering mechanisms mayorient or direct the energy application device 12 towards the targettissue 43 (or structure), and the controller 16 may then focus theenergy application onto the region of interest 44. The ultrasoundtransducer 74 may include an imaging transducer 74A used for spatialselection of the region of interest 44 within the target tissue 43. Theultrasound transducer 74 may include a treatment transducer 74B thatapplies the neuromodulation energy.

In some embodiments, an ultrasound image may be used to guide theultrasound stimulus to spatially select a region of interest fortargeted delivery of ultrasound stimulus. As provided herein, spatialselection or spatially selecting may include obtaining an image of atissue or organ (or a portion of a tissue or organ) and, based on image(e.g., the ultrasound image), identifying a region of interest withinthe organ. In some embodiments, the tissue or organ may have anatomicalfeatures that are used to guide the selection of the region of interestwithin the organ. Such features may, in some embodiments, include a siteof blood vessel or nerve entry into an organ, a tissue type within anorgan, an interior or edge of an organ, or a suborgan structure, by wayof non-limiting example. In certain embodiments, the anatomical featuremay include a liver porta hepatis, suborgans of a gastrointestinal tract(stomach, small intestines, large intestines), a pancreatic duct, or asplenic white pulp. By identifying the anatomical features in the image,the region of interest may be selected to overlap with or include theanatomical feature or be adjacent to the anatomical feature. In otherembodiments, the anatomical feature may be excluded from the region ofinterest. For example, an intestinal tissue may be selected as a regionof interest rather than a stomach tissue. The identification of theanatomical feature may be via morphological features that are visible inthe image (e.g., visible in the ultrasound image) or by structurerecognition features of the imaging modality used to obtain the image.As disclosed herein, the system 10 may be configured such that theenergy application device 12 is configured to operate in an imaging modeto obtain the image and to subsequently operate in energy applicationmode after the image is obtained and the region of interest is spatiallyselected based on the image.

In other embodiments, the region of interest may be identified by thepresence or absence of one or more biological markers. Such markers maybe assessed by staining the organ or tissue and obtaining imagesindicative of the stain to identify regions of the organ or tissue thatinclude the biological marker/s. In some embodiments, the biologicalmarker information may be obtained by in vivo staining technologies toobtain location data of the biological marker/s in the tissue or organspecific for the subject in real time. In other embodiments, thebiological marker information may be obtained by in vitro stainingtechnologies to obtain location data for one or more representativeimages that is then used to predict the locations of the biologicalmarker/s within the subject's tissue or organ. In some embodiments, theregion of interest is selected to correspond with portion of the tissueor organ that are rich in a particular biological marker or that lack aparticular biological marker. For example, the one or more biologicalmarkers may include markers for neuronal structures (e.g., myelin sheathmarkers).

The region of interest in the organ or tissue may be spatially selectedbased on operator input. For example, an operator may designate theregion of interest on the obtained image by directly manipulating theimage (i.e., drawing or writing the region of interest on the image) orby providing image coordinate information that corresponds to the regionof interest. In another embodiment, the region of interest may beautomatically selected based on the image data to achieve spatialselection. In some embodiments, the spatial selection includes storingdata related to the region of interest in a memory and accessing thedata.

Once spatially selected, the system 10 is configured to apply energy tothe region of interest as provided herein.

Examples Targeted Physiological Perturbations

A GE Vivid E9 ultrasound system and an 11 L probe were used for theultrasound scan before neuromodulation started. A focal areacorresponding to an interior region of interest was labeled on animalskin. The HIFU transducer was positioned on the labeled area. Anotherultrasound scan was also performed using a smaller imaging probe (3S),which was placed in the opening of the HIFU transducer. The imaging beamof the 3S probe was aligned with the HIFU beam. Therefore, one couldconfirm that the HIFU beam was targeted at the region of interest usingan image of the targeted organ (visualized on the ultrasound scanner).

Animal Protocols

Adult male obese Zucker rats, 8 to 12 weeks old (250-300 g; CharlesRiver Laboratories), in accordance with conditions maintained at thesupplier to promote development of insulin resistance and hyperglycemia.The rats were fed a high fat diet. Neuromodulation using appliedultrasound energy was performed on the liver. The ultrasound applicationwas performed for 1 minute. Blood samples were collected 15 minutesafter the last ultrasound treatment to analyze changes in circulatingcatecholamine concentration (e.g., norepinephrine and dopamine).Terminal blood samples were collected 60-90 minutes after the lastultrasound treatment to analyze changes in circulating moleculeconcentration. Blood samples were stored with the anti-coagulant(disodium) EDTA to prevent coagulation of samples.

The protocol used for ultrasound neuromodulation may be as follows:

-   -   (A) Animals may be anesthetized with 2-4% isoflurane.    -   (B) The animals may be laid prone on a water circulating warming        pad to prevent hyperthermia during the procedure.    -   (C) The region above the targeted region of interest for        ultrasound stimulus (e.g., nerve of interest) may be shaved with        a disposable razor and animal clippers prior to stimulation.    -   (D) Diagnostic imaging ultrasound may be used to spatially        select the region of interest.    -   (E) The area may be marked with a permanent marker for later        identification.    -   (F) Either an FUS ultrasound probe or a LogiQ E9 probe may be        placed at the designated region of interest previously        identified by the diagnostic imaging ultrasound.    -   (G) An ultrasound pulse may then be performed with a total        duration of a single stimulus not surpassing a single 1-minute        pulse. Energies of the ultrasound pulses would not reach levels        associated with thermal damage and ablation or cavitation (e.g.,        35 W/cm²).    -   (H) A second 1-minute ultrasound pulse may be applied.    -   (I) The animal may then be allowed to incubate under anesthesia        for acute study (e.g., 1 hour) and kinetic study. After which        the animal is sacked and tissue and blood samples are collected.        An incision may be made starting at the base of the peritoneal        cavity extending up and through to the pleural cavity. Organs        may be rapidly removed and homogenized in a solution of PBS,        containing phosphatase (0.2 mM phenylmethylsulfonyl fluoride, 5        μg/mL of aprotinin, 1 mM benzamidine, 1 mM sodium orthovandate        and 2 μM cantharidin) and protease (1 μL to 20 mg of tissue as        per Roche Diagnostics) inhibitors. A targeted final        concentration of 0.2 g tissue per mL PBS solution was applied in        all samples. Blood samples were stored with the anti-coagulant        (disodium) EDTA to prevent coagulation of samples. Samples are        then stored at −80° C. until analysis.

Target Tissue Stimulation and Physiological Perturbation for Diagnosisof Glucose Metabolic Disorders

The present examples demonstrate a noninvasive method to achievephysiological perturbation to assess a metabolic dysfunction in apatient. The disclosed techniques provide advantages relative totechniques for assessing metabolic dysfunction that involve hours ofpatient time, including fasting, infusion, and follow-on response/blooddraw steps. For example, in assessment of insulin resistance in apatient according to certain procedures, the patient may undergo afasting period, which permits the patient's physiological system toachieve a setpoint with respect to a population. After fasting, thepatient may be administered glucose or a metabolic active compound(e.g., orally or injected). The patient's response is then measured overa period of time, often hours, by monitoring a change in blood glucoseand/or insulin or other hormone that supports changes in the bloodglucose concentration. If either fasting glucose or insulin isdetermined to be above a range (determine by measuring previouspopulation of subjects) then a subject may be diagnosed with diabetes. AGlucose Tolerance Test measures how fast a bolus of glucose is clearedfrom blood in a fasting state (compared to a population of subjects) andis used to diagnose diabetes in subjects with higher (but notpathologically high) fasting glucose concentrations. Clamp techniquesare used to quantify how a subject metabolizes glucose. Clamps mayinclude a hyperglycemic clamp (continuous infusion of glucose; quantifycapacity for insulin secretion) or hyper-insulemic clamp (continuousinfusion of insulin; quantify insulin resistance) which bring issuesrelating to cost of assessment, and over complexity affects use in theclinic.

The HOMA (Homeostatic Model Assessment) Calculator uses a mathematicalmodel to estimate insulin sensitivity and B-cell function from plasmainsulin and glucose concentrations. This interaction between glucose andinsulin in the basal state provides information relating to the balancebetween hepatic glucose output and insulin secretion, which ismaintained by a feedback loop maintained by the liver and B-cells of thepancreas, allowing for HOMA to serve as a surrogate measure of steadystate beta cell function (% B) and insulin sensitivity (% S), aspercentages of a normal reference population. The predictions used inthe model arise from experimental data in humans and animals.

The disclosed techniques may replace or be used in conjunction withother techniques for diagnosis of glucose metabolic disorders toeliminate or shorten fasting and follow-on assessment times. In oneembodiment, a modified HOMA calculator may be based on the disclosedneuromodulation-induced perturbations to more closely approximate thefasted state of a diabetic subject. That is, rather than requiring asubject to fast for a certain number of hours, a subject (e.g.,diabetic, normal, or unknown) may be perturbed via neuromodulation tolower circulating glucose into a state resembling a fasting state forthe purposes of assessing glucose response. Moreover, neuromodulationinduces a stabilization of glucose levels independent of the feedingstate, as indicated by the lack of fluctuation in glucose levels. Takentogether, this neuromodulation induced stabilization of glucose reducesthe potential confounding results derived from feeding state assessmentsand permits an assessment of insulin sensitivity in the absence offeeding-derived effects

Ultrasound stimulation was performed as provided herein and according tothe timeline shown in FIG. 9 to show induced targeted physiologicalperturbations relative to a control. FIG. 10 shows the results ofultrasound stimulation of the liver (porta hepatis) in obese Zucker ratsrelative to control rats that underwent sham ultrasound treatment. Theultrasound-treated rats were protected from the increase in circulatingglucose in the control rats, even as both groups of rats were housed inconditions associated with the development of insulin resistance andhyperglycemia. Accordingly, the results demonstrate that ultrasoundtreatment causes perturbations in hyperglycemic rat populations that aredetectable via changes in circulating glucose. FIGS. 11 and 12 show feedconsumption and weight were similar between the treatment and controlgroups. The weight gain in the treated animals was slowed relative tothe control group (FIG. 12) over the experimental timeline while thefeed consumption remained steady (FIG. 11).

As provided herein, liver neuromodulation causes alteration to glucosemetabolism to provide a controlled/precise stimulus and modify therelationship between organs to influence or change a current clinicalscore of homeostasis (i.e., HOMA score), as shown in FIG. 13. Thedisclosed techniques may be used to generate an index reflective ofneuromodulation and resultant perturbations. Typically, the HOMA scoreis indicative of a relationship between fasting glucose and fastinginsulin measures. Generally, higher HOMA scores are reflective ofelevated levels of insulin required for glucose disposal. As providedherein, changes in insulin and glucose concentration may be assessed inresponse to liver neuromodulation as part of a diagnostic protocol toderive a homeostasis index that may be used to diagnose and trackinsulin resistance, even in subjects with glucose levels in a normalrange or not undergoing a controlled diet regimen.

The HOMA model for steady-state is based on circulating glucose andinsulin levels through a population-based formula. The assumption is thesubject is fasted. Further, the typical assessment is based on“stimulated” responses such as the glucose clamp, insulin clamp orglucose tolerance test. In certain embodiments, neuromodulation replacesthe typical fasting and stimulation (e.g., glucose clamp, insulin clamp,glucose tolerance). In one embodiment, the present techniques may beused to replace the glucose and/or insulin infusion with neuromodulationsuch that the test may be performed without administration of glucoseand/or insulin. However, it should be understood that neuromodulationfollowed by administration of glucose and/or insulin is alsocontemplated. In another embodiment, the present techniques may be usedto eliminate fasting from measurement protocols (by usingneuromodulation) to provide immediate deviation from baseline organkinetics/interactions.

As provided herein, the present techniques may be used to build apopulation-based model of a relationship between changes in glucose andinsulin concentration responses to neuromodulation. The model may bebased on glucose-insulin relationships for a population of healthyindividuals perturbed to an approximated fasting state vianeuromodulation. In certain embodiments, the model may be further basedon glucose-insulin relationships for a population of individuals withvarious types of metabolic function perturbed to an approximated fastingstate via neuromodulation. The model may applied to glucoseconcentration and insulin concentration data acquired from aneuromodulated subject perturbed into an approximated fasting state togenerate a score, whereby the score is indicative of a level of insulinresistance in the subject.

In one embodiment, the FIG. 14 is a flow diagram of a method 90 showinga metabolic dysfunction assessment technique. The technique acquires abaseline concentration of glucose and/or insulin at step 92, which maybe at a time point immediately before (within 1-6 hours before) orconcurrently with initiation of neuromodulation of a region of interestin a target tissue (e.g., liver, GI, pancreas) at step 94. As notedherein, the subject may be monitored using a continuous glucose monitorsuch that glucose concentrations are available on an ongoing basis.Based on the observed perturbation, which may be reflected in observingchanges in glucose and/or insulin concentrations over time as a resultof the neuromodulation at step 96, the clinical condition of the patientmay be determined at step 98.

The present techniques may be used to codify subject populations basedon level of glucose and/or insulin changes after stimulation or todiagnose type of diabetes and/or level of resistance. For example,subjects may be categorized as “responders” or “strong responders” or“low responders” or “nonresponders” depending on the level of glucoseand/or insulin changes. For example, a nonresponder may exhibit a changein concentration that is less than a predetermined threshold relative toa baseline concentration while a responder experiences a change inconcentration that is greater than the predetermined threshold. In oneexample, a nonresponder is considered to exhibit a change in glucose ofless than about 5%, less than about 10%, or less than about 15% relativeto baseline. A responder is considered to exhibit a change in glucose ofgreater than about 10-15% relative to baseline, while a strong responderis considered to exhibit a change in glucose of greater than about 50%relative to baseline. The responsiveness may be at a time pointassociated with an expected change in a healthy or responsivepopulation.

FIG. 15 is an example of a neuromodulation response profile for a highlyinsulin-resistant animal subject given a diet that drives insulinresistance and high glucose levels. The profile shows glucoseconcentrations 100 over a time period of several days before initiationof neuromodulation, showing relatively stable and high glucoseconcentrations above a threshold, shown as glucose concentration of 225mg/dL. Neuromodulation is initiated at baseline 102, and the glucoseresponse to neuromodulation is tracked over time. The depicted subjectis characterized by a steep glucose decrease of long duration andmagnitude and without any fasting or glucose administration. As providedherein, the response profile may be assessed or characterized by one ormore measures of response. In one embodiment, a magnitude of a changefrom a peak baseline concentration 102 to a trough 106 may be assessed.Alternatively or additionally, the slope 104 may be assessed (e.g., asteep slope may be indicative of insulin resistance). Additional metricsmay include a slope from a trough 106 to a recovery peak 110, adifference between a baseline concentration and a first recovery peak110, a time to the first recovery peak 101, and/or an area under thecurve 112 relative to a setpoint. For example, an insulin-resistantsubject may have a relatively short time period of lower glucoseconcentrations below a predetermined threshold (e.g., lower than 225mg/dL, lower than 200 mg/dL) before recovery to concentrations above thepredetermined threshold, which is reflected in the area under the curve112 being below an empirically-determined threshold. The subject may bemonitored during normal activities, such as eating and sleeping, and thecharacteristic profile may be reflective of changes in response to theseactivities relative to baseline. While previous techniques may haveinvolved complex glucose administration response tracking over severalhours in additional to pre-test fasting, the present techniques permitobservation of changes over a relatively short period of time, which isless of a burden on the tested subject. Accordingly, in one embodiment,the subject is assessed using glucose concentration changes that areobserved within 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, two hours,or within one hour after neuromodulation to achieve an approximatedfasting state in the subject. Further, because glucose monitoring may beaccomplished using glucose monitors that permit subjects to beambulatory and outside of a clinical setting, the present techniquesprovide a more convenient and faster assessment of metabolic dysfunctionthat does not require fasting or administration of a glucose bolus.

FIG. 16 is an example of a neuromodulation response profile for aninsulin-resistant subject with a starting glucose concentration that islower than that of the subject of FIG. 15. The profile shows apre-treatment glucose concentration 124 above a threshold (e.g.,approximately 250 mg/dL) and a higher baseline concentration 122 that islowered to a trough 126 in response to neuromodulation and a recoverypeak 128 or an area under the curve 112 relative to a setpoint. Forexample, an insulin-resistant subject may have a relatively short timeperiod of lower glucose concentrations below a predetermined threshold(e.g., lower than 225 mg/dL, lower than 200 mg/dL) before recovery toconcentrations above the predetermined threshold. The response profileis of lower magnitude and duration while nonetheless showing a steepdecrease in glucose concentrations. Accordingly, in certain embodiments,a response profile as in FIG. 15 and as in FIG. 16 may both beclassified as being insulin resistant, although in different bands orcategories. FIG. 17 is a response profile of a subject with a startingglucose concentration 130 below diabetic levels and that shows little orno response to neuromodulation. Accordingly, the subject may beconsidered to be a nonresponder and may be classified differentlyrelative to the profiles in FIG. 15 and FIG. 16.

In another embodiment, the disclosed techniques may be used fordetection of differences in subject populations for a variety ofperturbations and their associated clinical conditions, and/or optimaltherapeutic regimen. The subject population differences may result incategorizing the subjects into one of several categories and initiatingtreatment options based on the categorization. For example, drugresponse may be correlated to a responsiveness (e.g., a degree ofperturbation) to neuromodulation. Further, the disclosed techniques mayprovide category information (insulin resistant, not insulin resistant,responder, nonresponder) as well as additional data as part of a reportto a caregiver.

Target Tissue Stimulation and Physiological Perturbation forIdentification of Pathogen or Toxin Exposure

As provided herein, a neuromodulation response may be indicative ofpathogen, toxin, or environmental exposure prior to the time-point whereeither the pathogen itself is detectable or disease symptoms manifest(i.e., fever, etc.). That is, the present techniques provide an improvedand more rapid assessment of pathogen exposure. Such assessments mayimprove outcomes for hospital-associated pathogens that, when detectedmore rapidly, may be more effectively contained. While certaintechniques that track immune or systemic responses require continuousmonitoring to detect exposure to a pathogen, the present techniqueseliminate the requirement for baseline monitoring by comparing changesbefore and after neuromodulation in the expression of certain markers.

FIG. 18 is a flow diagram of a method 150 showing an immune responseassessment technique for identifying pathogen exposure by assessingresponse to neuromodulation of a region if interest in an immune tissueor structure, including lymphatic organs, the lymph vessels that extendthroughout the body and provide flow and drainage, the spleen, lymphnodes, Peyer's patches, and accessory lymphoid tissue (including thetonsils and appendix).

The technique acquires a baseline concentration of one or more markers,such as immune markers, at step 152, which may be at a time pointimmediately before (within 1-6 hours before) or concurrently withinitiation of neuromodulation of a region of interest in a target tissue(e.g., spleen or immune tissue) at step 154. Based on the observedperturbation, which may be reflected in observing changes in one or moremarkers relative to baseline at step 156, a pathogen exposure may beidentified at step 158. The change in concentration of one or moremarkers in response to neuromodulation that are associated with pathogenexposure or a lack of pathogen exposure may be identified according tothe techniques provided herein (blood concentration, tissueconcentration, system changes identified by sensor and/or image data).

FIG. 19 shows tumor necrosis factor mRNA expression changes that areultrasound-dose dependent for animal subjects exposed to endotoxin orlipopolysaccharide (LPS) and treated with neuromodulating energy appliedto the spleen. High dose treated animals experienced a largest decreasein tumor necrosis factor mRNA expression relative to control. FIG. 20shows mRNA expression array data for a number of markers for control andLPS-exposed and high and low dose ultrasound treated animals. FIG. 21shows an example protocol for acquiring baseline and potentialpost-exposure data for assessing a change in response based toneuromodulation after exposure to a material or pathogen. A baseline orcharacteristic pre-exposure response profile may be acquired andcompared to a potential post-exposure response profile to assess changesdriven by exposure.

The disclosed techniques as provided herein harness the targetedphysiological outcomes that are achievable via neuromodulation. Inaddition to implementations in which the targeted physiological outcomesare used to treat subjects, these outcomes may be used as part of adiagnostic protocol. For example, application of energy to a region ofinterest in a target tissue may result in predictable and targetedphysiological perturbations. One or more characteristics of theperturbations themselves or the subject's response to the perturbationsmay be indicative of a subject's clinical condition. For example, arapid return to homeostasis (e.g., return to baseline pre-modulationconcentrations of molecules of interest) within a pre-defined period oftime may be indicative of a healthy response to the perturbation. Incontrast, a slow response may be indicative of metabolic pathways thatare underperforming relative to those in a healthy subject. Whilecertain embodiments of the disclosure have been discussed in the contextof glucose regulation, it should be understood that the presenttechniques may be used to cause perturbations of other systems(including acute or chronic inflammatory conditions and related sensoryand neuroimmune effector systems) and, accordingly, to assess relatedclinical conditions.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims.

1.-8. (canceled)
 9. A method of inducing a physiological perturbation ina subject, the method comprising: directing energy of an energyapplication device at a region of interest of a subject; applying theenergy to the region of interest to induce a physiological perturbationas a result of the applying; assessing one or more characteristics ofthe physiological perturbation at one or more time points after theperturbation; and determining, based on the assessing, that the subjectis in a category selected from a first category or a second category,wherein the first category is responsive to neuromodulation and thesecond category is non-responsive to neuromodulation.
 10. The method ofclaim 9, wherein assessing one or more characteristics of theperturbation comprises: determining a concentration of a molecule ofinterest at a first time point after applying the energy; anddetermining a concentration of the molecule of interest at a second timepoint after applying the energy, the second time point being after thefirst time point.
 11. The method of claim 10, wherein assessing one ormore characteristics of the perturbation comprises determining adifference between the concentration of the molecule of interest at thefirst time point and the second time point, determining a slope of theconcentration of the molecule of interest between the first time pointand the second time point, or both.
 12. The method of claim 9, whereinassessing one or more characteristics of the perturbation comprisesdetermining a time between a trough and a recovery peak of a molecule ofinterest.
 13. The method of claim 9, comprising causing the energyapplication device to deliver treatment energy to the region ofinterest, wherein the treatment energy is applied according to atreatment protocol selected based on the clinical condition.
 14. Themethod of claim 9, wherein the region of interest is in a pancreas andwherein assessing the one or more characteristics comprises determininga concentration of circulating insulin at the plurality of time pointsafter the applying and comparing the concentration at the plurality oftime points to a baseline concentration of insulin to determine aresponsiveness of the subject to neuromodulation therapy, wherein thesubject is determined to be in the second category when a change in theconcentration relative to the baseline concentration is less than apredetermined threshold change.
 15. The method of claim 9, wherein theregion of interest is in a liver and wherein assessing the one or morecharacteristics comprises determining a concentration of glucose orinsulin at a plurality of time points after the applying and determiningthat a trend in the concentration at the plurality of time points isindicative of a return to a baseline glucose concentration within apredetermined time period.
 16. The method of claim 9, wherein thesubject is not fasted and wherein applying the energy causes anapproximated fasting state.
 17. The method of claim 9, wherein theregion of interest is in a spleen and wherein assessing the one or morecharacteristics comprises determining a change in a population of immunecells or concentration of cytokines or immune markers after deliveringthe energy.
 18. The method of claim 9, wherein the assessing the one ormore characteristics comprises receiving image data to identify asystemic response caused by the applying.
 19. (canceled)
 20. Amodulation system, comprising: an energy application device configuredto apply energy to a first region of interest in a first organ and to asecond region of interest in a second organ; and a controller configuredto: control a first application of the energy via the energy applicationdevice to the first region of interest to cause a first perturbation asa result of the first application of the energy; receive informationindicative of a first perturbation characteristic; control a secondapplication of the energy via the energy application device to thesecond region of interest to cause a second perturbation as a result ofthe second application of the energy; receive information indicative ofa second perturbation characteristic; determine a clinical condition ofthe subject based on the first perturbation characteristic and thesecond perturbation characteristic; and provide an indication of theclinical condition.
 21. The system of claim 20, wherein the firstperturbation characteristic comprises a glucose concentration at aplurality of time points after the first application of the energy tothe first region of interest.
 22. The system of claim 20, wherein thesecond application of energy to the second region of interest isresponsive to a determination that the first perturbation characteristicis associated with a first category.
 23. The system of claim 20, whereinthe first application of energy is before the second application ofenergy, concurrent with the second application of energy, or after thesecond application of energy.
 24. The system of claim 20, wherein thefirst perturbation characteristic and the second perturbationcharacteristic are combined to identify a combined response to determinethe clinical condition of the subject.
 25. The system of claim 20,wherein the first region of interest is in a liver and wherein thesecond region of interest is in a pancreas, and wherein the clinicalcondition is determined to be insulin resistance based on the firstperturbation characteristic being a decrease in glucose that is higherthan a predetermined glucose decrease threshold and the secondperturbation characteristic being an initial increase in circulatinginsulin that is higher than a predetermined insulin increase thresholdthat is followed by a subsequent decrease in the circulating insulinrelative to the initial increase.
 26. A method of assessing aphysiological perturbation in a subject, the method comprising: applyingultrasound energy to a region of interest in a subject to cause anapproximated fasting state in the subject via neuromodulation; receivingglucose and insulin concentration data from the subject in theapproximated fasting state; applying the data to a model, wherein themodel is based on a relationship between glucose and insulinconcentration in the approximated fasting state for a plurality ofnormal subjects; receiving an indication of an insulin resistance of thesubject using the model; and providing a treatment recommendation basedon the indication.
 27. The method of claim 26, wherein the treatmentrecommendation is an ultrasound treatment.
 28. The method of claim 9,wherein the controller is configured to control application of theenergy via the energy application device to the region of interest toinduce the physiological perturbation to cause a change in geneexpression, tissue displacement, tissue size changes, cell migration, orany combination thereof.
 29. The method of claim 28, wherein thephysiological perturbation comprises the change in tissue displacement,wherein the first category is based on the tissue displacement beinggreater than a predetermined threshold and the second category is basedon the tissue displacement being less than the predetermined threshold.30. A method of inducing a physiological perturbation in a subject, themethod comprising: directing energy of an energy application device at aregion of interest of a subject; applying the energy to the region ofinterest to induce a tissue displacement of the region of interest as aresult of the applying; acquiring image data at one or more time pointsafter the perturbation; measuring the tissue displacement using theimage data and image data acquired before application of the energy; andproviding an indication of a clinical condition of the subject based onthe assessing, wherein the indication comprises a determination that thesubject is in a category selected from a first category or a secondcategory based on the measured tissue displacement, wherein the firstcategory is responsive to neuromodulation based on the measured tissuedisplacement being greater than a predetermined threshold and the secondcategory is non-responsive to neuromodulation based on the measuredtissue displacement being less than the predetermined threshold.